CN117679420A - Application of interferon-induced gene pathway activation small molecule compound in hepatitis B treatment - Google Patents
Application of interferon-induced gene pathway activation small molecule compound in hepatitis B treatment Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
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Abstract
The invention discloses a small molecule C 19 H 14 F 3 N 3 O 2 Use of a S compound or a pharmaceutically acceptable salt thereof in the manufacture of an HBV inhibitor or a medicament for the treatment of a hepatitis b virus infectious disease or disorder; also disclosed is a pharmaceutical composition for inhibiting HBV replication or treating hepatitis B virus infectious diseases or disorders, which contains a compound represented by formula I or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable carrier. The invention screens out interferon inducible gene pathway activation micromolecule C from a compound library based on STAT1 double-luciferase reporter gene report experimental results 19 H 14 F 3 N 3 O 2 S, proved by experiments, has good inhibitionThe HBV transcription activity is prepared, and the method has wide application prospect in the aspect of hepatitis B treatment.
Description
Technical Field
The invention relates to the technical field of medicinal chemistry, in particular to application of an interferon-induced gene pathway activation small molecule compound in hepatitis B treatment.
Background
Hepatitis b virus (Hepatitis B Virus, HBV) infection is a major cause of chronic liver disease worldwide. According to World Health Organization (WHO) data, 2.96 million people in 2019 have been infected with chronic hepatitis b, about 150 ten thousand new infection cases per year, and about millions of people die due to cirrhosis or hepatocellular carcinoma, etc. caused by chronic HBV infection. Currently, 9000 thousands of chronic hepatitis B patients are estimated in China, wherein 2800 thousands of patients need treatment, and 700 thousands of patients need emergency treatment due to serious liver diseases and cancer incidence risks. Under the background, the traditional Chinese medicine composition can effectively treat the symptomatic patient and prevent the disease progress of the symptomatic patient, and is a main task of current hepatitis B prevention and treatment.
Currently, the primary drugs for treating patients with chronic hepatitis b include Interferons (IFNs) and nucleoside analogues (Nucleotide analog, NAs). Nucleoside analogs are effective in inhibiting viral replication, but difficult to clear HBsAg and cccDNA. And rebound after drug withdrawal, long-term administration is often required to maintain the effect, but viral resistance is easily caused. Adverse drug reactions were evident in patients following interferon treatment, and only 5-20% of patients achieved a sustained virologic response. The antiviral treatment of interferon for patients with combined liver function impairment can aggravate the liver function impairment degree of patients, and the liver function decompensator needs to avoid the application of interferon. Both of these drugs hardly realize "functional cure" of hepatitis b, i.e., elimination of hepatitis b surface antigen in a limited course of treatment. Therefore, the development of novel anti-HBV drugs is still a problem to be solved.
The interferon is an antiviral drug with limited treatment course, mainly induces the generation of interferon stimulating genes and acts on important biological processes such as HBV replication, transcription and the like through links such as encoding multi-antiviral proteins by an interferon signal path. Signaling and transcriptional activator proteins (signal transducer and activator oftranscription, STAT) are a family of proteins that bind DNA. STAT1 of the STAT family is an essential component of interferon signaling and is an important intracellular signaling factor. In the JAK-STAT classical signaling pathway, IFN is secreted from cells and binds to the receptor IFNAR1/2, further activating the signaling and transcriptional activators STAT1 and STAT2, phosphorylated STAT1 and STAT2 bind to IFN regulator 9 (IRF 9) to form heterotrimeric IFN regulator 3 (ISGF 3), ISGF3 enters the nucleus and binds to ISG regulatory elements (ISREs), activating transcription of interferon inducible genes (ISGs) to exert antiviral effects.
Disclosure of Invention
One of the objects of the present invention is to provide, in view of the above problems, the use of a compound represented by formula I or a pharmaceutically acceptable salt thereof for preparing an HBV inhibitor or a medicament for treating a hepatitis B virus infectious disease or disorder,
in the technical scheme of the application, the compound shown in the formula I or pharmaceutically acceptable salt thereof inhibits the transcription of the hepatitis B virus.
In the technical scheme of the application, the compound shown in the formula I or the pharmaceutically acceptable salt thereof reduces the expression level of HBV total RNAs, HBVpgRNA and HBV nucleoprotein.
In the technical scheme of the application, the compound shown in the formula I or pharmaceutically acceptable salt thereof reduces the generation level of hepatitis B HBeAg.
In the technical scheme of the application, the medicine further comprises a pharmaceutically acceptable carrier.
It is still another object of the present invention to provide a pharmaceutical composition for inhibiting replication of HBV or treating hepatitis B virus infectious diseases or disorders, comprising the compound represented by the above formula I or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable carrier.
In the technical scheme of the pharmaceutical composition, the active ingredient plays roles in reducing the generation level of HBeAg and inhibiting the transcription of hepatitis B virus.
In the technical scheme of the pharmaceutical composition, the product dosage form of the pharmaceutical composition is capsules, tablets, pills, paste, granules, oral solution preparations, microcapsule preparations or injection.
It is a final object of the present invention to provide the use of the above pharmaceutical composition for the preparation of an HBV inhibitor or for the preparation of a medicament for the treatment of a hepatitis b virus infectious disease or disorder.
In the use of the above pharmaceutical composition, the active ingredient in the pharmaceutical composition reduces HBeAg production level and inhibits hepatitis b virus transcription.
The beneficial effects of the invention are as follows:
screening interferon inducible gene pathway activation small molecule C from a compound library based on STAT1 dual-luciferase reporter gene report experimental results 19 H 14 F 3 N 3 O 2 S, and explore C 19 H 14 F 3 N 3 O 2 S in hepatitis b. Research shows that small molecule C 19 H 14 F 3 N 3 O 2 S can effectively reduce the expression level of HBeAg in cell supernatant, total HBVRNAs, HBVpgRNA in cells and HBV core protein, has good HBV transcription inhibition activity and has wide application prospect in the aspect of hepatitis B treatment.
Drawings
FIG. 1 shows a flow chart of the luciferase reporter system experiment (A) and the results of the STAT1 dual-luciferase reporter system verification experiment (B).
FIG. 2 shows the selection of small molecule C from a library of compounds 19 H 14 F 3 N 3 O 2 S.
FIG. 3 shows a different concentration gradient C 19 H 14 F 3 N 3 O 2 Study data of the effect of S treatment on HBV transcription levels in HepG2-NTCP cells statistical junction: a: ELISA analysis of different concentrations of C 19 H 14 F 3 N 3 O 2 S effect on HBeAg secretion; b: RT-PCR detection of different concentrations of C 19 H 14 F 3 N 3 O 2 S affects the expression level of total HBVRNAs in HepG 2-NTCP; c: RT-PCR detection of different concentrations of C 19 H 14 F 3 N 3 O 2 S effect on HBVpgRNA expression level in HepG 2-NTCP; d: noutblendon detection of different concentrations of C 19 H 14 F 3 N 3 O 2 S effect on HBVRNA levels; e: western blot detection of different concentrations of C 19 H 14 F 3 N 3 O 2 Effect of S on HBc expression.
Detailed Description
The invention is further illustrated, but is not limited, by the following examples.
The experimental methods in the following examples are conventional methods unless otherwise specified; the chemical and biological reagents used, unless otherwise specified, are all conventional in the art.
The reagents and materials used in the examples of the present application are mainly:
pGL3-Basic vector: promega corporation (USA);
luciferase detection reagent II (LAR II): the Promega company (usa) dual luciferase reporter system;
library of compounds: medChemexpress (MCE) company (U.S.).
Experimental example 1 construction of Dual luciferase reporter System test
The experimental flow of the construction of the dual-luciferase reporter gene system is shown in figure 1A, and the promoter region of the target gene STAT1 is inserted into a luciferase reporter gene vector pGL3 to construct a luciferase reporter gene plasmid. HepG2-NTCP cells were co-transfected with Renilla luciferase gene (Renilla) plasmid, and after 24 hours of transfection, the medium was removed, washed 2 times with PBS, and the PBS was blotted off. Then 250. Mu.L of 1 XPLB lysate was added to each well and incubated on a shaker for 15min at room temperature. And adding 50 mu L of luciferase detection reagent II (LAR II) into 10 mu L of cell lysate, blowing and mixing uniformly, and immediately detecting and reading by using a fluorescence illuminance instrument to obtain the value of firefly luciferase (Firefly luciferase). Then 50. Mu.L of substrate Stop & Glo of Renilla luciferase (Renilla luciferase) was added, annihilating the firefly luciferase reaction, and simultaneously activating the Renilla luciferase reaction. And (5) immediately detecting again by using a fluorescence illuminometer after blowing and mixing uniformly, and obtaining the value of the Renilla luciferase. The relative luciferase activity of the promoter can be obtained through data processing.
The STAT1 promoter region sequence is segmented and truncated (2000 bp,2500 bp), and is respectively inserted into a reporter gene vector pGL3 to construct a luciferase reporter plasmid, and the promoter activity of the luciferase reporter plasmid is detected. The reporter vector pGL3-Basic, which does not carry the promoter of interest, is the experimental control. Different fragment lengths of STAT1 recombinant plasmid and control plasmid were transfected into HepG2-NTCP cells and the relative luciferase activity of the different treatment groups was examined. As can be seen from the results of fig. 1B: compared with a control group, the relative luciferase activity of different truncated regions is increased to a certain extent, which indicates that the region plays an important role in STAT1 promoter activity, and the STAT1 dual-luciferase reporter gene report system is successfully constructed and can be used for subsequent experiments.
The nucleotide sequence of 2000bp truncated by segments of the STAT1 promoter region sequence is shown below (SEQ ID NO. 1):
TCATCTTGAATGATAACTCCCACAATTCCCACCTGTCATGGGAGGAAACTGGTGGGAGGTGATTAAATTATGGGGGCGAGACTTTCCTGCGCTGTTTTTGTGATAGTGAATGAGTCTCGAGGATCCGATCATTTTAAAAATGGGAGTTTCCCTGCACAAGCTCTTTGCCTGCCGCCATCAATGAAAGATGTGACTTGTTCCTCCTTGCCTCCCGCCATGATTGTGAGGCCTCCCCAGCCATGTGGAACTGTAAGTCCATTAAACCTCTTTATTTTGTAAATTGCCCAGTCTCAGGTCTTTATTAGCAGCGTTAAAAATGGATTAATACAGCTTTTTTCAGAGAACAGTAAAACATTGTCTTGGTTCTTGGGTGAGTAAGAGCAGAAGGGACACTTTTGGTGCCGTGGCATCTCTTGGCAGAGAAGTCTAATTTCACTGCTAACTTGCTTTGTGAGGGTGCATTTGTGACAAGAAGAACAAATAGATGTGCAAAGAAAAAAAACTCATACACACACATATAGGCATATCCATGGGAAATTATTCAGATCCTTCTCCTACCCCAAAGTGCCTAATTATTCAAGGCTTGACTCAAATCCCGTCTACTTGAAGGAACTAATTGATTTTATGAGCATATAGTCCAAAAAGATTTAGTTTGTCTGGCTTCAGCAATGCCTACCTGGAGAAGTGACATTTAAGCTGAGATTTTTAAACTAAGATCTAAAAGATGAGTTAGCCAGCGAAGAGTTGGGTGAAGCAGATGTTGGCTCAAGGGGTTTTCCAAACTCAGGGTGAGAAGTCCCTGAGTTTGGAGAGTGTGGTTTATTTGAAGAACTAAATGCTGAAGTAAGCAAAGCAGAGTGGCACATTATGGGGTTGGACAAAGAGATCGGGCAACCAGGGCTTTATAGTATGTATTAAGAAACTGAATTTTATAAGAGCAAAGAAAAGCCATGGAAGGATTTTCAGGTTGGCAAATGACATGATCTGCTTTTCAATTAAAAAAAAAAACACTCAAACTCTTGTATGGAGAATGGATTAAAAGGTAGATAAGGGCTGAAGTGAGGCAGCCATTCGGGAATCTACTGCAGAAGGAGGCAGCAGAAGAGATGGAGCAGAGTAAATGAGACTTGGGAAATATTTAGGAGGAAGAATCAATTAAGATTTGAGGATCAGGTGTTGCGAGGTGAGGGAGAAGGCAATGTCAACTATGAATCCTAGGTATCTGTCATGTGCCATTTCTAGAAAAAGAGCAGGTTTGAGGAGAAAGATGTTTGGTTCTGAACATAGTTTGTAGAGCTCACTGCACATACAAGTGGAGAGGCAAGTGGGAGTTGTAGGTGTGAAGCCCAGAGGAGAGGTGTGGACGGGATAAGCATTTAAGACTCCTCCATCTAGAAGGAAACTGAAGCTGTGGGTGAGGTCATCACAGCACAGCGTTTAGGAGAAGCCCAGGTAAAGAAGCTGACGAATGTCTGGACCCTGACAACCTTAACATATAATGGTTTGATAGTGGAGGTGGAGGCAATGTAGAAAGAATGCCAGAGGCAGGAAAAAGCAAGAAGGATGTGTTATCATCATGACCAAGGAAGAAACGTGTTTCAAGAACAAAGGCGTCAACTCTGCCCCATGCTTCCGAGCTGTCAAGTAAAGTGAGAAAAACAGAAAAGCGTTCCCTGGGTTTAGCAACACGGAGGTCAGTTGCTAAAGGGAGCTTCTAGAATGACGACGTCGCCAAATCTGTCCTCTGCCTGGATTCTCGGCGATGAAACTACTACAGAGACCTCCAAGTTTGGGCTTCTGCAAACACAGCACGTCCTTCTGATCGTTCTCTAAGATATGTAAACAGAACGCCAGTTCCCAGCGTGGCAACACGGGACTGGGCTGCAGCTCACCCAGCCGGCGGCCCCCGCCGGAAGCCGGCGGAAATACCCCAGCGCGTGGGCGGAGCAGCGGCCCGCAGAGGGAGGCGGTGGCGCCCACGGAACAGCCGCGTCTAATTG。
the nucleotide sequence of 2500bp obtained by sectioning and truncating the STAT1 promoter region is shown in the following (SEQ ID NO. 2):
CAATGATAGAAAAGGCTGGAAAGAAGGAAAGAAACATTTTGTGGAGGATCCAGACTAGCAGACAACATTTGTGTTTATCATATGTGCAAGGAGAGTTGTTAAGGCTTTTGAGCAGGGAATGACTATATCAATGCAGTATTTTAAGGAATTGAATTTGGCAAATATAGTGAAGGAGAAAGAGACAAGAAAAAGGTAGTATACCTAGAGATTAGCATAGCATTCCAATTCTCAAGTGATGAAGGCTTATGGTAGGTATTGCTGTGGATGCTAAAATAAAGGGGGGTAGGGAAGGAAGCATCCACAGATCTTGATGATCCAAATTGGGATTTGGAGTGCCAAATAATCCTATTTGCAGATGTTAGGATTATTCCATTCCTTCATCAATATTGAAGCTAATATCTGTGCAAGTTTTATAGTACCAGAAAAAAAATTTTTAACATGTAATCTACCCTTCCTAGAGTCAACTTTTGATATAGTTTGGCTGTGTCCCCACCCAAATCTCATCTTGAATGATAACTCCCACAATTCCCACCTGTCATGGGAGGAAACTGGTGGGAGGTGATTAAATTATGGGGGCGAGACTTTCCTGCGCTGTTTTTGTGATAGTGAATGAGTCTCGAGGATCCGATCATTTTAAAAATGGGAGTTTCCCTGCACAAGCTCTTTGCCTGCCGCCATCAATGAAAGATGTGACTTGTTCCTCCTTGCCTCCCGCCATGATTGTGAGGCCTCCCCAGCCATGTGGAACTGTAAGTCCATTAAACCTCTTTATTTTGTAAATTGCCCAGTCTCAGGTCTTTATTAGCAGCGTTAAAAATGGATTAATACAGCTTTTTTCAGAGAACAGTAAAACATTGTCTTGGTTCTTGGGTGAGTAAGAGCAGAAGGGACACTTTTGGTGCCGTGGCATCTCTTGGCAGAGAAGTCTAATTTCACTGCTAACTTGCTTTGTGAGGGTGCATTTGTGACAAGAAGAACAAATAGATGTGCAAAGAAAAAAAACTCATACACACACATATAGGCATATCCATGGGAAATTATTCAGATCCTTCTCCTACCCCAAAGTGCCTAATTATTCAAGGCTTGACTCAAATCCCGTCTACTTGAAGGAACTAATTGATTTTATGAGCATATAGTCCAAAAAGATTTAGTTTGTCTGGCTTCAGCAATGCCTACCTGGAGAAGTGACATTTAAGCTGAGATTTTTAAACTAAGATCTAAAAGATGAGTTAGCCAGCGAAGAGTTGGGTGAAGCAGATGTTGGCTCAAGGGGTTTTCCAAACTCAGGGTGAGAAGTCCCTGAGTTTGGAGAGTGTGGTTTATTTGAAGAACTAAATGCTGAAGTAAGCAAAGCAGAGTGGCACATTATGGGGTTGGACAAAGAGATCGGGCAACCAGGGCTTTATAGTATGTATTAAGAAACTGAATTTTATAAGAGCAAAGAAAAGCCATGGAAGGATTTTCAGGTTGGCAAATGACATGATCTGCTTTTCAATTAAAAAAAAAAACACTCAAACTCTTGTATGGAGAATGGATTAAAAGGTAGATAAGGGCTGAAGTGAGGCAGCCATTCGGGAATCTACTGCAGAAGGAGGCAGCAGAAGAGATGGAGCAGAGTAAATGAGACTTGGGAAATATTTAGGAGGAAGAATCAATTAAGATTTGAGGATCAGGTGTTGCGAGGTGAGGGAGAAGGCAATGTCAACTATGAATCCTAGGTATCTGTCATGTGCCATTTCTAGAAAAAGAGCAGGTTTGAGGAGAAAGATGTTTGGTTCTGAACATAGTTTGTAGAGCTCACTGCACATACAAGTGGAGAGGCAAGTGGGAGTTGTAGGTGTGAAGCCCAGAGGAGAGGTGTGGACGGGATAAGCATTTAAGACTCCTCCATCTAGAAGGAAACTGAAGCTGTGGGTGAGGTCATCACAGCACAGCGTTTAGGAGAAGCCCAGGTAAAGAAGCTGACGAATGTCTGGACCCTGACAACCTTAACATATAATGGTTTGATAGTGGAGGTGGAGGCAATGTAGAAAGAATGCCAGAGGCAGGAAAAAGCAAGAAGGATGTGTTATCATCATGACCAAGGAAGAAACGTGTTTCAAGAACAAAGGCGTCAACTCTGCCCCATGCTTCCGAGCTGTCAAGTAAAGTGAGAAAAACAGAAAAGCGTTCCCTGGGTTTAGCAACACGGAGGTCAGTTGCTAAAGGGAGCTTCTAGAATGACGACGTCGCCAAATCTGTCCTCTGCCTGGATTCTCGGCGATGAAACTACTACAGAGACCTCCAAGTTTGGGCTTCTGCAAACACAGCACGTCCTTCTGATCGTTCTCTAAGATATGTAAACAGAACGCCAGTTCCCAGCGTGGCAACACGGGACTGGGCTGCAGCTCACCCAGCCGGCGGCCCCCGCCGGAAGCCGGCGGAAATACCCCAGCGCGTGGGCGGAGCAGCGGCCCGCAGAGGGAGGCGGTGGCGCCCACGGAACAGCCGCGTCTAATTG。
example 2 screening of Small molecule C from a library of Compounds 19 H 14 F 3 N 3 O 2 S
First, 2.8X10 5 Individual/well human hepatoma HepG2-NTCP cells were seeded in 12-well plates. After 24h, the cells were treated by changing to PMM medium (hepatocyte maintenance medium) and adding different compounds, respectively. After 24h treatment, the cells were incubated with a mixture of infection medium (Williams E medium, 10% foetal calf serum, 1% penicillin-streptomycin mixture, 1% L-glutamine and 2% DMSO), 4% PEG8000 and HBV concentrate (infection factor 1000 genome equivalent) in an incubator at 37℃for 24h. The PBS buffer was rinsed 4 times and replaced with infection medium, placed in an incubator at 37℃for a corresponding period of time, and the medium was replaced every other day.
And screening interferon-induced gene pathway activation small molecules from a compound library, wherein the primary screening adopts an ELISA method to screen the influence of the compound on the secretion of HBeAg in the supernatant of the HepG2-NTCP cells. The above HepG2-NTCP cell treatment was used to detect the secretion level of HBeAg in the culture supernatant of 7d cells according to the ELISA kit (Kovar. Kei, china). Using HBeAg inhibition fold change > 30% as the selection threshold, we identified 45 compounds. The secondary screening uses 45 compounds identified previously, each of which was treated with the aforementioned HepG2-NTCP cells, and the expression level of total HBV RNAs/HBV pgRNA in the cells was detected by RT-PCR. Using HBV RNA inhibition fold change > 50% as the selection threshold, we identified 6 compounds.
The effect of the compound on STAT1 promoter activity was again screened using STAT1 dual luciferase reporter gene system. After 48h of treatment of HepG2-NTCP cells with the compounds to be screened, the relative luciferase activity of the different treatment groups was examined. It was artificially prescribed that a compound which had not caused significant cell death by visual observation under a microscope, which had increased the relative luciferase activity by 2-fold or more as compared with the negative control to which DMSO alone was added, was used as a candidate compound. The experimental results show that the compound C 19 H 14 F 3 N 3 O 2 The relative ratio of luciferase in the S treatment group is obviously increased compared with that in the control group, and the ratio is increased by more than 2 times. And C 19 H 14 F 3 N 3 O 2 S inhibits HBV RNA levels and HBeAg levels in a dose-dependent manner.
Wherein, the primer sequence of Real-time PCR is as follows:
PCR reaction system: total volume: DNAmasterMIX 5. Mu.L, template cDNA 1. Mu.L, upstream and downstream primers 0.5. Mu.L each, the balance ddH 2 O. PCR reaction conditions: 95 ℃ for 10min;95℃10s,60℃30s,72℃15s, for a total of 35 cycles.
Based on the results of the three screening experiments, select C 19 H 14 F 3 N 3 O 2 S was used as candidate compound for further investigation and C was explored 19 H 14 F 3 N 3 O 2 S is in BThe underlying mechanism of action in hepatitis.
C 19 H 14 F 3 N 3 O 2 The Chinese name of S is 5- (3, 4-dimethoxy phenyl) -2- (thiophene-2-yl) -7- (trifluoromethyl) pyrazolo [1,5-a]Pyrimidine with CAS number 883003-62-1 has chemical structural formula as shown in formula I:
example 3C 19 H 14 F 3 N 3 O 2 Study of the Effect of S on HBV transcription level in HepG2-NTCP cells
First, 2.8X10 5 Individual/well human hepatoma HepG2-NTCP cells were seeded in 12-well plates. After 24h, the culture medium was changed to PMM medium (hepatocyte maintenance medium) and C was used with different concentration gradients (0, 10, 20 or 40. Mu.M) 19 H 14 F 3 N 3 O 2 S treated cells for 24h, respectively. HBV virus was diluted in medium containing 4% peg8000 (1000 infection of multiple HBV virus per cell) and then added to each well of 12-well plate and incubated in incubator at 37 ℃ for 24h. The PBS buffer was rinsed 4 times and replaced with infection medium, placed in an incubator at 37℃for a corresponding period of time, and the medium was replaced every other day.
The secretion level of HBeAg in the culture supernatant of 7d cells was detected according to the ELISA kit (Kovar. Kei, china) protocol. As can be seen from the results of fig. 3A: c at different concentrations 19 H 14 F 3 N 3 O 2 S significantly inhibited secretion of HBeAg.
The aforementioned HepG2-NTCP cell treatment, followed by 7d treatment, extraction of intracellular total RNA using Trizol reagent, thereby detecting C 19 H 14 F 3 N 3 O 2 S effects on expression of HBV pgRNA and total HBV RNAs, primer sequences of RT-PCR, PCR reaction system and PCR reaction conditions were the same as in example 2. From the results of fig. 3b, c, it can be seen that: RT-PCR analysis confirmed different concentrations of C 19 H 14 F 3 N 3 O 2 S treatment is obviousThe expression level of total HBV RNAs and HBV pgRNA in HepG2-NTCP cells is inhibited. Further detecting different concentrations of C by a Northern blot method 19 H 14 F 3 N 3 O 2 Effect of S on HBV RNA levels. As can be seen from the results of fig. 3D: c at a different concentration than the control group 19 H 14 F 3 N 3 O 2 S treatment can obviously reduce the production of 3.5, 2.4 and 2.1kb HBV RNA in cells.
The aforementioned HepG2-NTCP cell treatment, followed by extraction of total cell proteins after 7d treatment, and detection of C 19 H 14 F 3 N 3 O 2 Effect of S on HBV protein expression. As can be seen from the results of fig. 3E: westernblot results show different concentrations of C 19 H 14 F 3 N 3 O 2 S can significantly inhibit HBV core (HBc) protein level.
Experimental study of the invention shows that small molecule C 19 H 14 F 3 N 3 O 2 S can effectively reduce the expression level of HBeAg in cell supernatant, intracellular HBV total RNAs, HBV pgRNA and HBV nucleoprotein, has good HBV transcription inhibition activity and has wide application prospect in the aspect of hepatitis B treatment.
Claims (10)
1. The application of the compound shown in the formula I or pharmaceutically acceptable salt thereof in preparing HBV inhibitor or medicine for treating hepatitis B virus infectious diseases or symptoms,
2. the use according to claim 1, characterized in that: a compound of formula I or a pharmaceutically acceptable salt thereof inhibits hepatitis B virus transcription.
3. The use according to claim 2, characterized in that: the compound of formula I or a pharmaceutically acceptable salt thereof reduces the expression level of HBV total RNAs, HBVpgRNA and HBV ribonucleoprotein.
4. The use according to claim 1, characterized in that: the compound shown in the formula I or pharmaceutically acceptable salt thereof reduces the production level of hepatitis B HBeAg.
5. The use according to claim 1, characterized in that: the medicament also comprises a pharmaceutically acceptable carrier.
6. A pharmaceutical composition for inhibiting HBV replication or treating a hepatitis b virus infectious disease or disorder, characterized in that: comprising as active ingredient a compound of formula I according to claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
7. The pharmaceutical composition according to claim 6, wherein: the active ingredient acts by reducing HBeAg production levels and inhibiting hepatitis b virus transcription.
8. The pharmaceutical composition according to claim 6, wherein: the pharmaceutical composition is in the form of capsule, tablet, pill, paste, granule, oral solution, microcapsule or injection.
9. Use of the pharmaceutical composition of claim 6 for the preparation of an HBV inhibitor or for the preparation of a medicament for the treatment of a hepatitis b virus infectious disease or disorder.
10. The use according to claim 9, characterized in that: the active ingredients in the pharmaceutical composition reduce HBeAg production levels and inhibit hepatitis b virus transcription.
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| CN112055749A (en) * | 2018-04-05 | 2020-12-08 | 豪夫迈·罗氏有限公司 | Use of FUBP1 inhibitors for the treatment of hepatitis B virus infection |
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| CN102178927A (en) * | 2011-03-01 | 2011-09-14 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Application of interferon-induced transmembrane protein 3 (IFITM 3) for preparing medicament against hepatitis B virus (HBV) infection |
| CN112055749A (en) * | 2018-04-05 | 2020-12-08 | 豪夫迈·罗氏有限公司 | Use of FUBP1 inhibitors for the treatment of hepatitis B virus infection |
Non-Patent Citations (1)
| Title |
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| 陈仁;廖金瑶;陈文莉;黄晶;马晓军;罗晓丹;: "干扰素诱导蛋白10在慢性乙型肝炎病毒感染中的临床作用分析", 中华医院感染学杂志, no. 15, 8 August 2016 (2016-08-08), pages 3358 - 3359 * |
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